Salmonella is an important foodborne pathogen that causes enteric disease in a variety of hosts. Symptoms of Salmonellosis include diarrhea, mild fever, nausea, and abdominal pains with the symptoms normally developing within 12-72 hours after consumption of contaminated food, particularly raw or undercooked foods such as meat, eggs and dairy products.
The genus Salmonella is comprised of two species, Salmonella enterica and Salmonella bongori (Brenner et al. 2000). Salm. enterica is further divided into six subspecies (I, II, IIIa, Mb, IV and VI) with S. enterica subspecies I comprising of the majority of clinically relevant serotypes. Although illnesses because of subspecies I are more prevalent, infections involving other subspecies (IIIa, Mb, IV and VI) and Salm. bongori are also sporadically seen (Chong et al. 1991; Snehalatha et al. 1992; Nastasi et al. 1999; Ma et al. 2003; Mahajan et al. 2003). Non-typhoidal salmonellosis is usually self-limiting and does not require antibiotic therapy in healthy individuals. However, treatment is necessary in immunocompromised individuals, children and the elderly. Rapid detection of the organism is therefore essential for faster diagnosis and treatment. Recent Salmonella outbreaks linked to ready-to-eat foods necessitate rapid detection methods (CDC 2008, 2009) as conventional Salmonella identification methods can be laborious and time-consuming, sometimes taking between 4-7 days to complete.
Real-time PCR has been used in the past for Salmonella detection because of its rapidity, sensitivity and accuracy (Rodriguez-Lazaro et al. 2003; Csordas et al. 2004). Several studies have successfully targeted invA gene for the detection of Salmonella species by real-time PCR (Cheng et al. 2008; Gallegos-Robles et al. 2009). However, as invA gene is a virulence gene located on the Salmonella pathogenicity island 1 (SPI 1) acquired by horizontal gene transfer, it may be genetically unstable or absent in some serotypes including Salmonella serotype Senftenberg (Ginocchio et al. 1997). Therefore, it is prudent to include targets in addition to invA for rapid molecular detection of Salmonella species by real time PCR.
A number of studies have tested other genes including iroB (Baumler et al. 1997), hilA (Ziemer and Steadham 2003), pipA, sopE, sopB and mgtC (Khoo et al. 2009). However, these targets failed to detect either Salm. bongori (iroB and hilA) (Baumler et al. 1997; Ziemer and Steadham 2003) or certain serotypes of Salm. enterica subspecies I (pipA sopE, sopB, mgtC) (Khoo et al. 2009).
A recent study has tested putative type III secretion ATP synthase gene (ssaN) and found it to demonstrate 100% inclusivity for the 40 Salmonella strains (S. enterica subspecies) tested and 100% exclusivity for 24 non-Salmonella strains after a 6 hour incubation period. (Chen et al. 2010) Other recent studies have tested the hilA gene for detection of Salmonella species and found that hilA was capable of detecting 30 S. enterica serovars but not the 30 non-salmonellae strains (McCabe E. et al., Validation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica serovars, Journal of Microbiological Methods, 2011, 84:19-26). A continuation of this study found that hilA was capable of detecting 106 Salmonella enterica subspecies enterica strains but not the 30 non-salmonellae strains (McCabe E. et al., Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica, Food Microbiology, 2011, 28:447-456). The results of the above mentioned studies are limited to the Salmonella enterica subspecies. No other strains, subspecies or serotypes of Salmonella were investigated.
Given the shortcomings of the prior art, what is needed is a rapid, sensitive method of detecting all subspecies of Salmonella using a gene specific to Salmonella. 